Project Summary/Abstract Multiple Myeloma (MM) causes devastating bone destruction and protracted suppression of bone formation that markedly increases fracture risk, impacts mortality and contributes to MM cell drug resistance. MM bone lesions rarely heal in most patients, so that severe bone pain and fracture risk persist. Currently, there are no safe bone anabolic agents approved for MM bone disease (MMBD). We showed that the multi-domain protein p62 (sequestosome-1) is a major contributor to MMBD through its two major functions. It is a signaling hub for multiple signaling pathways that increase MM cell growth, bone destruction OB suppression in MM. p62 also mediates pro-survival functions in MM cells as a cargo receptor for ubiquitin-mediated autophagy. The p62-ZZ domain (p62-ZZ), plays a key role in both these functions, and is the focus of this proposal. We, with colleagues at University of Pittsburgh, developed XRK3F2 (XRK), a small molecule that binds p62-ZZ. We found that XRK induced dramatic new bone formation in MM-bearing mice, allowed MM patient-derived bone marrow stromal cells (MM-BMSC) to differentiate to functional OB, induced MM cell death and enhanced the anti-MM effects of Bortezomib (Btz) and Carfilzomib, proteasome inhibitors (PIs) used for MM treatment. However, the mechanisms responsible for XRK?s effects in MM are unclear. Recently, our collaborators found that p62-ZZ is a high-affinity N-recognin for N-terminal argininylated proteins generated by the N-end rule pathway, and that XRK acts as a high affinity degron for p62- ZZ, mimicking endogenous N-end degrons. The N-end rule pathway (N-ERP) is a proteolytic system that protects cells from the proteotoxic stress of misfolded proteins (MPs) in the cytosol that normally are targeted for ER-associated degradation, and shuttles them to proteasome and autophagy-mediated degradation pathways. Excess MPs trigger release of chaperones from the ER that are then N-arginylated and bind MPs. The N-R protein complexes bind either the UBR box of N-recognins for subsequent proteosomal degradation, or p62-ZZ for autophagic proteolysis. We recently found that saturating p62-ZZ with XRK increased PI-induced MM cell death via necroptosis, even in PI-resistant cells. Thus, p62-ZZ serves as the molecular switch for necroptotic vs. apoptotic cell death pathways in MM cells. However, how XRK?s actions as a high-affinity N-degron that binds p62-ZZ are uncharacterized in MM or BMSCs. We will use in vitro and in vivo approaches to determine; Aim 1: The mechanisms responsible for XRK?s relief of the suppressed OB differentiation in BMSC exposed to MM cells or TNF?, and the contributions of inflammatory cytokines to this process. Aim 2: The mechanisms responsible and relative contributions of XRK?s effects on osteocytes in MMBD. Aim 3: The importance of necroptosis-mediated cell death in MM, and its regulation by the p62-ZZ/N- ERP and proteasome pathways. Aim 4: The in vivo effects of combining XRK targeting of p62-ZZ +/- PI treatment on MMBD and healing MM bone lesions. These studies should provide important new information on the p62- ZZ/N-ERP pathway as a therapeutic target to build bone and decrease tumor growth in MMBD.